It is often desirable to detect very small amounts of nucleic acids, such as samples obtained from biological samples. According to one common approach, nucleic acids, target nucleic acids, are extracted from the sample and are hybridized to an oligonucleotide to form a detectable complex. In order to obtain a detectable signal that can be correlated with the amount of the target, either the target nucleic acid or the oligonucleotide is associated with a signal generating reporter element, such as a radioactive atom, a fluorescent or chromogenic molecule, or an enzyme which is capable of converting a substrate into a product which can be detected and measured. The signal generated, directly or indirectly, by a properly hybridized nucleic acid is detected and measured by methods known in the art.
Many of the commonly used techniques of molecular biology, including fractionation and identification of specific sequences of nucleotide bases, involve the immobilization of target nucleic acid sequences on solid supports. For example, target nucleic acid sequences have been immobilized on nylon or nitrocellulose membranes, then detected with an oligonucleotide having a radioactive label attached thereto. Alternatively, so-called "sandwich" hybridization systems may be employed, using a capture oligonucleotide, which includes a sequence of nucleotide bases homologous to or complementary to the target and which is covalently attached to, or non-covalently associated with, a solid support, and using a detection oligonucleotide, which is an oligonucleotide covalently attached to, or non-covalently associated with, a reporter group, such as a radioactive label or a detection enzyme, and that has a sufficient complementarity with the target nucleic acid sequence in a region that is different from that portion of the target nucleic acid sequence which hybridizes to the capture oligonucleotide under conditions such that a dual hybridization occurs with the target sequence. The capture oligonucleotide, target nucleic acid and detection oligonucleotide form a sandwich complex by hybridization of the target with both the capture and detection oligonucleotide.
The solid support may be in the form of beads in which case the assay is referred to as a "bead-based sandwich hybridization system" (herein referred to as a BBSHS). A BBSHS is described, for example, in European Patent Application No. 276,302. According to this method, in a first step, the target nucleic acid and an oligonucleotide probe used for its detection, which is complementary to a first region of the target, are hybridized. The complex thus formed is then captured by a second oligonucleotide that is complementary to a different region of the target. In addition, the capture oligonucleotide is preferably end-attached to a solid support. The amount of the detection oligonucleotide associated with the solid support after these hybridization steps is directly related to the amount of the target nucleic acid captured. In this way, the BBSHS can be used to determine the amount of a specific single-stranded nucleic acid in a sample. In this and similar assays, radioactively (e.g., .sup.32 P) labeled cloned DNAs or synthetic oligonucleotides are most commonly employed because of the high sensitivity which can be obtained with such labels. .sup.32 P-labeled oligonucleotide probes used in conjunction with SEPHACRYL.TM. or TRISACRYL.TM. (Sepracor Inc.) beads in BBSHS experiments provide about 10:1 or better signal to noise ratios with target sequences present in about 0.5 mole amounts.
In practice, because of the inconveniences associated with handling, storage and disposal of radioisotopes, non-radioisotopic reporter systems are often used. Successful application of a non-radioisotopic reporter system requires a detection system which exhibits high sensitivities and low background properties when used in conjunction with the reporter system on a given solid support. The SEPHACRYL.TM. bead supports have been previously shown to possess serious limitations when used in conjunction with non-radioisotopic (e.g., colorimetric) detection systems (see, for example, International Application No. PCT/US90/00089). For example, the colorimetric signal from enzyme-oligonucleotide conjugates in sandwich formats and direct capture experiments on Sephacryl beads are compromised by undesirable background signal level, thereby giving low signal to noise ratios.
Non-specific background (in the presence of target nucleic acid) can be a result of:
1) hybridization of the detection and capture oligonucleotides to non-exact sequences of the target nucleic acid; PA0 2) direct hybridization of the detection oligonucleotide to the capture oligonucleotide; or PA0 3) non-specific attachment of the detection oligonucleotide to the bead support or walls of the reaction vessel.
While the first two of these possible cause as can be minimized by sufficiently stringent solution hybridization, capture and wash conditions, non-specific binding properties are poorly understood.
As described in International Application No. PCT/US90/00089, TRISACRYL.TM. support has been shown to be a more selective support than SEPHACRYL.TM. for use in bead-based sandwich hybridization assays. When Trisacryl is used, however, in a bead-based hybridization assay with non-isotopic detection, e.g., using alkaline phosphatase as the reporter enzyme and a chemiluminescent (herein abbreviated as CL) substrate, the sensitivity of the Trisacryl-based assay is only about one femtomole (see, e.g., the Examples provided herein).
It would be desirable to find solid supports that have better binding properties, including reduced levels of non-specific attachment of the oligonucleotides used for detection of the target nucleic acids and increased capture potential of the immobilized probe, especially when used in conjunction with non-radioisotopic detection systems.
Desirable properties of solid supports contemplated for use in hybridization detection of nucleic acids include: hydrophilicity; ease of handling, including the ability to form stable suspensions thereby obviating the need for agitation of the assay mixture, as well as compatibility with standard recovery techniques, such as filtration or centrifugation; suitable functional groups on the surface of the solid support; and low non-specific binding with the detection oligonucleotides.
Typical solid supports employed for direct capture and sandwich hybridizations are, for example, nitrocellulose or nylon membranes, activated agarose supports and diazotized cellulose supports. These supports, however, do not meet all of the above criteria. For example, the bonds between these supports and the capture oligonucleotides-are either not covalent, thereby allowing a certain release of the oligonucleotides from the support, or the supports have other shortcomings. For example, N-hydroxysuccinimide or cyanogen bromide activated polysaccharide affinity supports have a serious drawback in the leakage of ligands, which interferes with affinity purification. If the free ligand that leaks from the support is more effective as a binder than the insolubilized ligand, the free ligand binds the target macromolecule essentially irreversibly, and prevents affinity adsorption to the column. Further, cyanogen bromide activation of polysaccharide supports leads to the formation of N-substituted isoureas on the surface of the matrix. These confer undesirable ion exchange properties to the support, which become problematic in affinity chromatography, when analytes (such as nucleic acids) are present in very minute concentrations.
Therefore, solid supports with cross-linked, polymeric matrix structures, to which capture oligonucleotides can be covalently and stably end-attached, and which meet the above criteria, are continually being sought for improved hybridization assay systems.
Therefore, it is an object of this invention to provide solid supports with cross-linked, polymeric matrix structures, to which capture oligonucleotides can be covalently and stably end-attached for use in such sandwich hybridization systems and assays.
It is also an object to provide sandwich hybridization assay systems and assays that are sufficiently sensitive to detect very low concentrations of a target nucleic acid and that may be sufficiently sensitive to distinguish between target nucleotides that differ in sequence in only a few nucleotides.